Assessment of Iris albicans lange as potential antimicrobial and analgesic agent

Objective of the present work was to evaluate the presence of phytochemical constituents and pharmacological activities (antimicrobial and anti-nociceptive) of crude extract isolated from Iris albicans and its corresponding fractions. Extraction was made with methanol and extract was evaluated for the presence of different bioactive constituents, as per standard protocols. Extract and its corresponding fractions were evaluated for their antimicrobial and anti-nociceptive potential. Well diffusion method was used to evaluate the antibacterial effects while anti-nociceptive effect was studied using in-vivo models (acetic acid induced writhing, hotplate and tail immersion tests) at different dose level (100, 200 and 300 mg/kg of body weight) and compared with diclofenac sodium (dose = 10 mg/kg body weight). Results showed that the I. albicans extracts contained secondary metabolites including alkaloids, phytosterols, flavonoids, saponins, terpenoids, tannins, phenols, steroids, fixed oil, glycosides and carbohydrates. Furthermore, it was observed that different fractions in decreasing polarity order such as chloroform >n-Hexane > Ethyl acetate > Crude Methanol > Aqueous extract exhibited effective antimicrobial response against all test organisms. Results of the study showed that the extracts have significant antimicrobial and analgesic activity, providing evidence for its folklore use.


Introduction
Antibiotics resistance is a global issue which lead to severe health hazards and compelling the researchers to either bring structural modification in the existing antibiotics or to develop natural alternative antimicrobial products [1]. Several species of ornamental plants have been considered for their tremendous antimicrobial activity [2,3]. In South Asia, above 80% of the population depends on traditional medicines and local healers as the primary source of treatment [4]. This is mainly due to easy accessibility and affordability of consulting with traditional medical practitioners [4,5]. In Pakistan, herbal drugs have been traditionally used for the treatment of pain, inflammation and infectious diseases for many decades [3]. However thorough evaluation is needed to scientifically prove their safety and efficacy. (3.20 Kg) was crushed to coarse powder (3.08 Kg), soaked in methanol (15 L) for two weeks at room temperature and filtered. This process was repeated three times. The extract was concentrated through rotary evaporator at 40 ± 2˚C. For complete removal of methanol dried extract was kept at 40˚Ć on water bath until the extract was completely dried. The dried crude extract was then subjected to fractionation [10,11].

Fractionation of crude extract
Sequential fractionation of the crude extract was performed using solvents of different polarity [12]. Fraction was started with solvents of low polarity and ended with highest polarity i.e. nhexane > chloroform > ethyl acetate>aqueous. The isolated fractions were concentrated by rotary evaporation. The concentrated fractions were dried completely by evaporation of organic solvents on water bath at 40 ± 5˚C and water was evaporated by keeping the concentrated fraction in hot air drier. The dried fractions were kept in a tightly closed container, properly labelled and reconstituted with different solvent on need basis for different experiments.

Dose selection and grouping
For determination of anti-nociceptive activity, animals were distributed in five groups, each comprising of six animals (n = 6). Animals with similar age were placed in same group and age of treatment and control group were kept similar i.e., animal with a similar age were kept in same group. Normal saline was used as negative control while diclofenac sodium (Wellmark Pharma, Pakistan; 10 mg/kg of body weight; administered by intra peritoneal route) was used as positive control [13]. Animal groups were treated with 100 mg/kg body weight, 200 mg/kg body weight and 300 mg/kg body weight doses of extract and solvent fractions. Doses were selected on the basis of reported literature [13] to find out response at different level of dose.

Phytochemical screening
Crude extract of iris albicans and its corresponding fractions (IACCF) were evaluated for their phytochemical constituents using standard protocols [13][14][15]. These protocols and procedure were related to identification of phyto-constituents like phenolic compounds, glycosides, alkaloids, saponins, terpenoids, flavonoids, anthraquinones, steroids and tannins as described [14,15]. Test for carbohydrates. Extract was soaked in purified water (5 mL) for 5 min and filtered. Alcoholic α-naphthol solution (3 drops) was added to filtrate, taken in a test tube and observed the junction. Presence of carbohydrates was confirmed by appearance of violet ring at the junction [14,15].
Test for alkaloids. Presence of alkaloids in the eaxtract was confirmed by Mayer's test. Extract (0.5 g) was extracted with methanol and added HCl (2N). Heated the mixture at 40 ± 3˚C, cooled and filtered it. This filtrate was reacted with Mayer's reagent (potassium mercuric iodide) and checked the precipitate growth. Appearance of yellow color precipitate confirmed alkaloids presence [14,15].
Test for saponins. Extract was soaked in boiling water in a test tube. After specified time, mixture was cooled and vigorously shaken till froth formation. Afterward the test tube was placed in stand for~15 min and noted the results. Strongly positive (+++) meant more than 5 cm froth, (++) meant more than 2 cm froth, (+) meant <1 cm froth and (-) represent no froth [14,15].
Test for phenols. The extract was soaked in water and filtered. Filtrate was reacted with ferric chloride solution (3-4 drops) and observed formation of precipitate. The appearance of bluish black color precipitate indicated presence of phenol [14,15].

Anti-microbial assay
Anti-microbial assay involved determination of both anti-bacterial and anti-fungal activity. Both the assays were performed by well diffusion method [16], using dimethyl sulphoxide (DMSO) as solvent and agar was used as growth media. Bacterial and fungal strains were obtained from Department of Microbiology, Abasyn University Peshawar, Pakistan. Expert from department of Microbiology confirmed their identity and purity as per USP-38/NF-33 protocols [17]. All the strains were kept in air tight containers, under standard conditions of storage as specified by USP 38-NF 33. Media was evaluated for its suitability for the growth of the selected strains of bacteria and fungi as per protocols for anti-microbial assay of United States Pharmacopeia [17]. During determination of suitability of media for microbial growth, different parameters for incubation like incubation time and temperature, were also optimized. The selected. Growth media was taken in plates and inoculated with selected strains of bacteria (P.aeruginosa, E.coli, S. typhi, S. epidermis, S. aureus, C. freundii, A. israelii, Y. enterocolitis.) and fungi (A. nigre, A. flavus, P. notatum), separately. Sterile borer (diameter = 6 mm) were used to make wells in the developed agar plates. Solution of test extract (100 mg/mL) was prepared in DMSO and each test extract (10 μL) was added in to separate well (1 mg / well). Meropenem (10 μg/disc) and levofloxacine (5 μg/disc) acted as positive control for bacterial culture plates while Nystatin (100 units/disc) and fluconazole (25 μg/disc) were used as positive control for fungal culture plates. In both the cases DMSO was used as negative control and its final volume was 100 μL. Bacterial culture plates were incubated at 37 ± 2˚C for 24 h while fungi culture plates were incubated at 24 ± 2˚C for 48-72 h. After completion of incubation period, zone of inhibition was measured around each well [18] from bottom of the plate.

Anti-nociceptive activity
Anti-nociceptive activity of the extracts was evaluated by; • Acetic acid induced writhes test • Hot plate method

• Tail immersion method
Acetic acid induced writhes test. During the test, pain was initiated by intra peritoneal administration of acetic acid and treated with diclofenac sodium (10 mg/kg body weight; reference drug / positive control) and different doses (100, 200 and 300 mg/kg body weight) of test extracts from I. albicans. Crude extract and its fractions were administered orally by a gastric tube, about 30 minutes prior to initiation of experiment. Acetic acid solution (0.7%) was administered by intra peritoneal route (10 mL/kg body weight) to initiate pain (writhing response/count) in test animals. Then after 1 h, writhing initiated by the acetic acid were counted (for 30 min), after 5 min of latency period. The mice were treated with diclofenac sodium (standard drug/ positive control), normal saline (negative control; 10 mL/Kg body weight) and different doses of test extracts of I. albicans (100, 200 and 300 mg/kg body weight). Normal saline was used as solvent for preparation of solutions of plant extracts and dilution of diclofenac sodium injection. Oral administration of extracts and positive control was carried out through gastric tube and animals were properly acclimated to the procedure. Normal saline was used as negative control and was administered orally. The test animals were placed under constant surveillance. The total numbers of writhing were counted for 20 min. The percentage of anti-nociceptive effects was ascertained by following equation as described [18,19].
Hot plate method. The design of hot-plate method was described [20,21] with slight changes in the context of present study. Mice were restrained from food overnight with readily access to water. Apparatus used resembled UGO hot-plate BASILE 7280, Germany, with some modifications, employed for analgesic activity of diclofenac sodium (10 mL/Kg body) and the IACCF. Hot-plate temperature was kept at 55 ± 0.5˚C and mice were placed on the hot-plate. Mice response to radiant heat induced analgesia stimulus and paws licking or jumping were noted. These parameters were used as an indication or mice's latency of analgesia responses. Paws licking or jumping time for each mouse was noted (reaction time) and 45 s was set as cut-off period avoiding damage to the limbs and paws. Only animals were selected showing jumping or licking reactions within the range of 15 s for current study (24 h) preceding to the initiation of experiment. Mice function as their own control as well as response time was calculated in triplicate preceding treatment; by 1 h interval. Each of the mice in the group was thereafter treated with a solution of IACCF (dose 100 mg/Kg, 200 mg/Kg and 300 mg/Kg) of each of the five extracts was prepared in normal saline.
The reaction times of all mice were individually re-evaluated after 0, 30, 60, 90, 120 and 180 min of treatment. At last each treatment group's test mean was calculated. Following treatment response or reaction time was represented by this mean (Ta) as well as specified percentage of thermal stimulus of pain by utilizing the below mentioned formula;

Thermal Stimulus percent of Protection ¼ Mean of Test ðTaÞ À Mean of Control ðTbÞ Mean of Control ðTbÞ ð2Þ
Tail immersion method. The lower 3 cm part of mice tail was appropriately marked and immersed in water bath at 55 ± 1˚C. After a few seconds, the mice responded by flicking the tail. It is the reaction time (in 0.5-2 s units by a stopwatch). After each value the tail was dried carefully. For the test and standard drugs given orally reaction time was recorded prior and after administration periodically and 15 second was set up the cut off time. Test animals were adults Swiss albino mice. Test animals weighing between 25-29 g were utilized for tail immersion activity. Albino mice were scheduled in six groups. A solution of I. albican (dose-100 mg/ kg/10 μL, 200 mg/kg/10 μL, 300 mg/kg/10 μL) of each of the five extracts was formed in N/S. Test-1: solution of I. albican (1-dose-100 mg/Kg.bw/10 μL, 200 mg/kg/10 μL, 300 mg/kg/ 10 μL) of each of the five extracts, Test-2: A solution of diclofenac sodium (10 mg/Kg of body weight) was prepared. After 60 min of oral administration of test and standard drugs, the observations were recorded in the interval of 0, 30, 60, 90, 120 and 180 min [19]. Like hot-plate method thermal pain stimulus calculated by following formula: Statistical analysis. The results were analyzed by statistical means utilizing one-way ANOVA followed by Dunnet's t-test, using the software "Graph Pad Prism" version 5 (San Diego, CA, USA). P (0.05) was considered as significant value. Results were presented as Mean ± SEM.

Phytochemical screening of Iris albicans
In the current study, methanolic extract of I. albicans and its corresponding fractions were screened for the presence of secondary metabolites like flavonoids, glycosides, terpenoids, alkaloids, steroids, saponins and reducing sugar. Methanolic extract of I. albicans and its corresponding fractions displayed the presence of all active metabolites as shown in Table 1, It confirms importance of the extract from I. albicans with respect to the presence of medicinally important constituents.

Biological evaluation of I. albicans
Crude extract of I. albicans and its corresponding fractions were evaluated for antibacterial, antifungal assay through standard protocols.
Evaluation of anti-bacterial activity. The I. albicans and its corresponding fractions were evaluated through well diffusion method against eight bacterial strains (staphylococcus aureus, escherichia coli, salmonella typhi, staphylococcus epidermidis, pseudomonas aeruginosa, citrobacter freundii, actinomycetes israelii and yersinia enterocolitica). Mereopenem and levofloxacin were used as standard (reference) drugs. All samples were effective against pseudomonas aeruginosa while comparing with the standard levofloxacin, ethyl acetate fraction showed moderate activity against all test organisms except staphylococcus epidermidis. The results obtained for samples against citrobacter freundi and actinomycetes israelii were almost same and significant compare to both standards used while zone of inhibition of samples against salmonella typhi and staphylococcus aureus were same and moderately effective. Results of antibacterial activity of I. albicans extract and its sub fractions are presented in Table 2.

Figs 1 and 2 represents zone of inhibition of extracts against specific bacteria.
Anti-fungal activity of I. albicans. The crude and fractions were subjected to antifungal assay using nystatin and fluconazole as standard drugs. Fungal strains used in the study included aspergillus niger, aspergillus flavus and penicillium. The crude and corresponding fractions were effective against A. niger while DMSO was used as solvent and was inactive against all fungal strain as shown in Table 3.
Comparison of anti-fungal effect produced by the crude extracts and its sub fractions is presented in Fig 3.

In vivo biological activity of I. albicans
Analgesic activity. I. albicans has been traditionally used for their analgesic and anti-gout effect which makes basis for evaluation of its anti-nociceptive potential. Analgesic activity was based on acetic acid induced writhe's test, hot-plate test and Tail-immersion test.
Acetic acid induced writhing test. The effect of I. albicans and its corresponding fractions on the writhing response in mice is shown in Table 4. The plant extract and its fractions numerically (P = 0.05) prevented the acetic acid induced effects and highest inhibitory response (41.72 to 60.51 as compared to control) was observed at highest dose (300 mg/kg body weight). The inhibitory potential of I. albicans and its corresponding fractions at 300 mg/kg was better but in some cases was lower than diclofenac sodium at a dose of 10 mg/Kg (62.8%). All fractions displayed analgesic activity but chloroform and n-hexane showed significant inhibition. In the pursuing of acetic acid test the extracts of s I. albicans and its corresponding fractions showed numerically significant activity. The order of inhibition is; Chloroform>n-Hexane>Ethyl acetate>Crude Methanol>Aqueous. It was found that less hydrophilic extracts, showed better analgesic activity in comparison with more hydrophilic fractions. Higher dose (300 mg/kg) of all extracts showed better inhibition of writhes in mice while lower doses (200 and 100 mg/kg) of all extract fractions, except chloroform and n-hexane displayed moderate effects.
Hot-plate test in mice. The findings of the hot-plate test showed that the IACCF has potential anti-nociceptive effect in relation to control groups. The IACCF in doses of 100, 200 and 300 mg/kg of body weight were effective and comparable with diclofenac sodium injection, (10 mg/kg), as shown in Table 5. No adverse effects seen with I. albicans extract and no animals died of IACCF treatment.
Comparison of analgesic effect of the crude extract and its sub-fractions is presented in Fig 4. Tail-immersion test in mice. In the tail immersion test all fractions displayed analgesic activity. However, chloroform and n-hexane showed maximum inhibition followed by ethyl acetate, crude and aqueous. In tail immersion test; the extracts of iris albicans showed numerically significant dose dependent activity i.e. 300 mg > 200 mg > 100 mg. It was also observed that lipid soluble extracts showed better activity as compared to the low lipid soluble extracts. At 300 mg/kg dose, all the extracts possessed excellent decrease in licking and jumping response in mice while 200 mg dose of all extracts except chloroform and n-hexane fractions; showed moderate effects. However; all extracts (100, 200 and 300 mg/Kg body weight) significantly increase latency period as shown in Table 6.

Discussion
The phytochemical analysis of I. albicans and its corresponding fractions showed the presence of different bioactive constituents (primary and secondary metabolites) including alkaloids, saponins, terpenoids, tannins, flavonoids, phenols, steroids, fixed oil, glycosides and carbohydrates. Presence of different bioactive constituents provides a scientific reason for the its flockier use.
I. albicans and its corresponding fractions have excellent antimicrobial effects against a wide range of microorganisms. I. albicans and its corresponding fractions showed excellent inhibition potential against bacterial and fungal species like escherichia coli, staphylococcus aureus, staphylococcus epidermidis, pseudomonas aeruginosa, salmonella typhi, citrobacter freundii, actinomycetes israelii, yersini, aspergillus niger, aspergillus flavus and penicillium. These antimicrobial results were unique and novel due to employing of well diffusion technique for crude extract of I. albicans and its corresponding sub-fractions. Furthermore, globally accepted solvents have been used during the study [4,22,23] minimizing the interference by the solvents.
The antimicrobial studies revealed that I. albicans and its corresponding fractions have the potential for the isolation and discovery of new drug products from plant. Chloroform extract showed same inhibitory potential as levofloxacin (zone of inhibition 24 mm). The sequential antimicrobial potential efficacy is as; Chloroform extract >n-hexane >methanol (crude) extract >ethyl acetate extract >aqueous extract  Comparison with reported literature showed that flavonoids, phenols, phtosterols and terpenoids were responsible for the antimicrobial activity of I. albicans and its corresponding fractions [2,3,9]. These compounds were copiously found in crude methanol, ethyl acetate as well as chloroform extracts. This study showed that IA it is a potential candidate for isolation of an effective anti-microbial drug.
The anti-nociceptive potential of I. albicans and its corresponding fractions were analyzed by; • Acetic acid induced writhes test • Hot plate method • Tail immersion test All the tests were performed according to the reported methods [24,25] mice models. These analgesic models were chosen to confirm authenticate and validate the possessing of both anti-nociceptive responses i.e., central and peripheral responses, so as to allow the evaluation of the expected mechanism of effect of constituent of the extract [26].
Being an irritating agent; acetic acid induces pain, with characteristic abdominal constrictions by stimulating local receptors of peritoneum [25]. Abdominal constrictions effect has been linked with presence of prostanoids e.g. raised levels of PG-E and PGF2α in the fluids of peritoneal and lipoxygenase products. These effects had been reported after the administration of acetic acid [27]. The acetic acid-induced test is very sensitive, simple and capable of detecting analgesia at lowest possible dose which is not seen with other methods and test, like the tail flick method [28]. The inhibitory effect exhibited by the extracts is proposed to have a peripherally mediated analgesic activity which may be linked to inhibition of lipo-oxygenases and cyclooxygenases [25]. Peritoneal administration of dilute acetic acid increases release of mediators which in turn activate the terminals of primary afferent fibers, preventing nociception  [29]. The multiple extracts caused a remarkable prolongation of response time to pain stimuli [30]. Centrally mediated action may likely be through opioid receptors in the CNS [30,31]. Opioid analgesics relieve pain by raising the pain threshold at the spinal cord level and by altering the brain's perception of pain. The ability of the extracts of Iris albicans to suppress pain perception in triplet (the acetic acid-induced test, the hot plate and tail immersion tests) suggests that the anti-nociceptive activity is possibly mediated by a series of mechanisms that comprises both central as well as peripheral pathways of pain intuition. Also glycosides, especially alcoholic glycosides, have proven analgesic and antimicrobial activities [3]. Constituents such as triterpenes and saponins produced anti-nociceptive actions on nervous system and cardio-vascular effects [32]. It may also contributed to the analgesic effect.

Conclusion
It was concluded from the study that I. albicans has many medicinally important phytochemicals like alkaloids, flavonoids, phtosterols, phenols, carbohydrates, terpenoids, tannins, and saponins, and has shown significant pharmacological activities such as analgesic effect, antibacterial effect and anti-fungal activity. This study will provide a scientific base for its medicinal use. Furthermore, it is suggested that I. albicans should be further processed for isolation, characterization and structure elucidation of bioactive compounds, responsible for pharmacological activities.

Limitations of the study
The study was conducted with objective to provide scientific proof for acute use of the extract from I. albicans. Some of the limitations of the study are; • The study provides results for acute use of the I. albicans extract and no data has been provided for chronic use of the extract.
• Qualitative characterization of individual fraction was not performed.

Future perspective
In the presented study only the acute effect of the plant extract has been studied. The effect produced by chronic use of the IA extract will be evaluated.